In modern in-vitro clinical diagnostics, a variety of methods are utilized for the detection of analytes in a sample. In one form of a diagnostic method, an immunoassay, one or more specifically binding species are used. Typical examples are the sandwich immunoassay, where two specifically binding species (antibody or antigen) bind to the analyte of interest, and the competitive immunoassay, where the analyte of interest and an analog of this analyte compete for binding to a specifically binding species. One of the specifically binding species is commonly attached to a so-called label or tag, which may be an atom (e.g., radioactive), molecule (e.g., an enzyme, fluorescent, or luminescent compound) or particle (magnetic or latex). This label allows for detection of the analyte of interest through a variety of detection methods corresponding to the label utilized. In a competitive assay, either the specifically binding species or the analyte analog can carry the label.
The other specifically binding species is frequently associated to a solid or suspendable substrate (“the solid phase”) covalently or through adsorption. Alternatively, it may be linked to a first member of a second binding pair (e.g., biotin), while the second member of the second binding pair (e.g., streptavidin) is attached to the solid phase. This allows the specifically binding species to bind to the solid phase via the second binding pair interaction (e.g., biotin-streptavidin).
Solid phases may be macroscopic solid phases, such as microtiter well, tube and ball in tube devices, or suspendable solid phases, such as beads, latex beads, magnetic latex beads, as well as other paramagnetic materials. A secondary binding species is commonly labeled through the use of a tag. The interaction between the tag and the solid phase allows for detection and quantification of the analyte of interest through a variety of detection methods corresponding to the label utilized.
It is important for the stability and reproducibility of the diagnostic assay that the binding ability of the solid-phase bound species for its binding partner does not become impaired over time. This may result in decreased reliability and decreased assay sensitivity. One mechanism that may lead to such impairment (apparent as instability) is bleeding of a portion of the solid-phase-attached species into the surrounding medium. The free species competes with the solid-phase-attached species for binding to the target and usually has a significant kinetic advantage due to its faster diffusion. Therefore, it is advantageous to maintain the amount of free species competing with the solid phase-attached species in a reagent constant, preferably very close to zero. This would allow for the sensitive detection of analytes in a stable and reproducible manner. The preferred way of eliminating free species is to find a binding method that will eliminate dissociation. Covalent bonding, as opposed to adsorptive association, might be the method of choice, due to its greater bond strength. However, in many cases this may be impossible or impractical, for various reasons.
For example, the material chosen for the solid-phase may lack suitable active groups for covalent coupling. If that is the case, due to the type of assay, or for cost considerations, adsorption will then be the preferred choice of association. An alternative method involves having a binding species that is attached to the solid-phase material indirectly through an auxiliary binding species. An example of this would be a biotinylated antigen or antibody that is attached to an avidin coated substrate, avidin being the auxiliary species. The auxiliary binding species itself can be associated with the solid phase via a covalent bond or by adsorption. This method may be utilized when the binding species becomes inactivated if directly associated with the solid-phase material. Again, since the affinity constant of the auxiliary binding species is not infinite, some dissociation, in the form of bleeding, may occur.
A further method includes binding species consisting of subunits which are either non-covalently associated with each other or have a covalent bond that is reversible under normal storage conditions. Subunits that are in this way reversibly and indirectly attached to the solid phase can disassociate from the surface-coupled subunit(s). If the free subunit retains its binding ability in the assay, or regains it after combining with other free subunits, the free subunit may interfere in the assay.
If bleeding cannot be eliminated, several practices, each with its particular disadvantages, have been employed to compensate for bleeding, including the following:
1) Frequent recalibration to correct for the drift caused by bleeding of the binding species. The most obvious disadvantage is additional cost for reagent and time spent on recalibration. The bleeding may also limit shelf life due to assay signals dropping to insufficient levels.
2) Supplying the immunoassays and other diagnostic assay reagents in a dried form, such as lyophilized or air-dried product. This approach usually eliminates bleeding, but dry reagents typically need to be reconstituted by addition of liquid, which requires time and effort by a user. The drying process itself requires additional processing by the manufacturer and introduces a source of inhomogeneity and bottle-to-bottle variation not found in reagents in a liquid form. The drying process may also increase lot-to-lot variation.
3) If the solid phase is stationary (e.g. microtiter plate) or can be conveniently separated from the liquid (magnetic beads or other, larger or smaller sized beads), it is possible to “wash” the reagent with water or an appropriate liquid before beginning the assay. A disadvantage of this approach involves the additional time, effort, and materials required for a “pre-wash” step and the limits imposed on flexibility in designing the assay sequence.
U.S. Pat. No. 5,212,063 discloses a ligand trap useful for reducing or eliminating biotin interference in immunoassays that employ biotin labeled antibodies (or other biotinylated species). Biotin, which naturally occurs in body fluids, can compete with the biotinylated species for binding sites on the avidin or streptavidin conjugate employed in these assays and cause erroneous results. Biotin is selectively removed from the immunological reaction by incubating the sample solution with polymer particles consisting of a streptavidin-modified core and a biopolymer coating.
U.S. Pat. No. 5,863,740 discloses an interference-eliminating agent through the modification and inactivation of Streptavidin molecules by biotin saturation, chemical modification or genetic engineering.
U.S. Pat. No. 4,256,834 discloses a fluorescent scavenger particle immunoassay. A reagent employed in the reaction contains a signal repressor. This signal repressor interacts with a label connected to a member of a signal producing system in bulk solution. Upon coupling of the label and member of the signal producing system to the homologous member of the signal generating system, the signal repressor no longer interferes.
If the attachment of the binding species to its solid phase is not irreversible, and the potential remedies discussed above are not an option, a certain amount of the binding species will be released into the fluid medium. This may lead to impairment of the assay, typically evidenced as instability.
The situation is further exacerbated because production of reagents typically occurs a significant amount of time prior to their actual use. This creates the necessity of storing the assay reagents under a variety of conditions. During storage and transport, there may be irregularities in temperature, which may detrimentally affect the release of solid-phase bound species. These variables of time, temperature and motion all contribute to the dissociation of the binding species from the solid-phase material.
Although dissociation will primarily take place prior to utilization of the assay reagents, the impairment will result when a sample, such as a bodily fluid, is added. The dissociated species will then compete with the solid-phase attached species for binding to the target and usually has a significant kinetic advantage due to its faster diffusion rate. When the amount of target is limited, even a small amount of free material can significantly reduce the number of targets bound to the solid phase. This, in turn, can yield false results that, even when detected, will require reruns and recalibration.
It is therefore highly desirable to keep the amount of free species in reagents constant, preferably very close to zero. When permanent association of the binding species to the solid-phase is physically impossible or economically prohibitive, a remedy must be provided to avoid instability that can result in incorrect test results.